(Circulation. 1995;91:1847-1854.)
© 1995 American Heart Association, Inc.
Articles |
Cholesterol Derivative of a New Triantennary Cluster Galactoside Lowers Serum Cholesterol Levels and Enhances Secretion of Bile Acids in the Rat
Presented in part at the 66th Scientific Sessions of the American Heart Association and in abstract form (Circulation. 1993;88:465).
From the Division of Biopharmaceutics, Leiden-Amsterdam Center for Drug Research, Sylvius Laboratory, University of Leiden (the Netherlands).
Correspondence to Erik A.L. Biessen, PhD, Division of Biopharmaceutics, Leiden-Amsterdam Center for Drug Research, Sylvius Laboratory, University of Leiden, PO Box 9503, 3200 RA Leiden, Netherlands.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Previous studies have demonstrated that cholesterol-derivatized galactosides exert a hypocholesterolemic effect by inducing hepatic uptake of atherogenic lipoproteins by means of galactose-recognizing receptors in the liver. However, a prolonged infusion of high concentrations of these compounds was required for this effect, possibly because of low affinity for the galactose-recognizing asialoglycoprotein receptor on the parenchymal liver cell.
Methods and Results We have designed a new series of
triantennary galactosides to optimize the affinity and specificity for
this receptor. The affinity of a triantennary galactoside for the
asialoglycoprotein receptor appeared to be dramatically enhanced by
proper spacing of the three terminal galactose groups. In rats, a
single injection of
N-[tris-O-(3,6,9-trioxaundecanyl-ß-D-galactopyranosyl)methoxymethyl]methyl-N
-[1-(6-(5-cholesten-3ß-yloxy)glycyl)adipyl]glycinamide
[TG(20Å)C], the cholesterol derivative of the most selective
galactoside, causes a dose-dependent decrease of 
45% in the serum
cholesterol concentration (P<.001). This decrease is mainly
attributed to a decrease in the level of serum HDL (P=.0066)
and, to a lesser extent, serum LDL (P=.036). In addition,
TG(20Å)C strongly enhances the bile-acid secretion in rats during the
first 2 hours after administration, which indicates that
TG(20Å)C-induced clearance of cholesterol from the bloodstream is
efficiently coupled to hepatic bile-acid secretion.
Conclusions We conclude that TG(20Å)C efficiently directs lipoproteins that contain cholesterol to the liver at a 30-fold-lower concentration than previously developed cholesterol-derivatized cluster galactosides. This newly developed approach to lower cholesterol levels may prove valuable for familial hypercholesterolemic patients or those with familial defective apolipoprotein B-100 who do not respond or who respond insufficiently, respectively, to conventional therapies.
Key Words: hypercholesterolemia • cholesterol • lipoproteins
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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High levels of serum LDL, the major vehicle for cholesterol transport in humans, are correlated with an increased occurrence of arteriosclerosis.1 2 Therapy for hypercholesterolemia is mainly focused on the induction of LDL receptors in the liver, which leads to an enhanced clearance and thus catabolism of LDL from the bloodstream. This process is accomplished through inhibition of cholesterol synthesis with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors or by stimulation of biliary cholesterol excretion with bile-acid sequestrants.3 4 However, patients with the homozygous form of familial hypercholesterolemia, who lack functional LDL receptors, do not respond to such therapy,5 whereas in patients with familial defective apolipoprotein (apo) B-100,6 7 the above therapy may be of limited value.8 Furthermore, blockade of cholesterol synthesis may influence the level of intermediates of the cholesterol synthesis pathway that are essentially involved in various cellular functions.9 These unwanted side effects are circumvented by a direct removal of LDL through an alternative high-capacity receptor on the parenchymal liver cell, the asialoglycoprotein receptor. This receptor is involved in hepatic uptake and subsequent lysosomal processing of galactose-terminated substrates.10 11 Theoretically, a bifunctional compound that consists of a lipophilic moiety for anchorage to LDL and a second moiety displaying a high affinity for the asialoglycoprotein receptor should enhance the catabolism of LDL. Earlier studies from our department12 13 14 15 indicated that use of this concept to lower serum lipoprotein levels is valid. In 1984, a cholesterylated cluster galactoside with hypocholesterolemic activity, N-[tris-O-(ß-D-galactopyranosyl)methyl]methyl-N
-[(5-cholesten-3ß-yloxy)succinyl]glycinamide
[TG(4Å)C], was synthesized.16 However, continuous
intravenous infusion of high doses of TG(4Å)C was required to
accomplish a detectable therapeutic effect.12
Subsequently, monogalactosylated cholesterol derivative, which
displayed a comparable hypocholesterolemic activity,15 was
synthesized.17 Both compounds stimulated the hepatic
uptake of LDL by directing LDL to the galactose-recognizing receptor on
the Kupffer cell.13 14 15 Both the low
level of
lipid-lowering activity and the lack of specificity for targeting
lipoproteins to the asialoglycoprotein receptor were argued to be
caused by the moderate affinity and specificity of the
cholesterol-derivatized cluster galactoside for this receptor.
In the present study, we show that both the affinity and
specificity of a triantennary cluster galactoside for the
asialoglycoprotein receptor could be significantly improved by
elongation of the spacer that connects the terminal galactose moieties
of a cluster galactoside with the branching point of the dendrite from
4 to 20Å. In view of its high affinity, the most selective compound,
N-[tris-O-(3,6,9-trioxaundecanyl-ß-D-galactopyranosyl)methoxymethyl]methyl-N-[1-(6-methyl)adipyl]glycinamide
[TG(20Å)], may offer a new tool for the development of a more
potent
hypocholesterolemic therapeutic treatment. Therefore, we synthesized
the cholesterol derivative of this cluster galactoside,
N-[tris-O-(3,6,9-trioxaundecanyl-ß-D-galactopyranosyl)methoxymethyl]methyl-N-[1-(6-(5-cholesten-3ß-yloxy)glycyl)adipyl]glycinamide
[TG(20Å)C], and tested its cholesterol-lowering activity and
its
effect on the biliary secretion of bile acids.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Reagents
Carrier-free sodium 125I was supplied by Amersham International. Collagenase type I, N-acetyl-D-galactosamine, and BSA (fraction V) were obtained from Sigma Chemical Co. All other chemicals were analytical grade. N-[tris-O-(ß-D-glucopyranosyl)methyl]methyl-N
-[4-O-(5-cholesten-3ß-yl)succinyl]glycinamide
[TGlc(4Å)C] was synthesized according to the procedure
described by
Kempen et al.16 The synthesis of
N-[tris-O-(ß-D-galactopyranosyl)methyl]methyl-N-[6-(1-O-methyl)adipyl]glycinamide
[TG(4Å)],
N-[tris-O-(ethyl-ß-D-galactopyranosyl)methoxymethyl]ethyl-N-[1-(6-methyl)adipyl]glycinamide
[TG(9Å)],
N-[tris-O-(propyl-ß-D-galactopyranosyl)methoxymethyl]methyl-N-[1-(6-methyl)adipyl]glycinamide
[TG(10Å)],
N-[tris-O-(3-oxapentyl-ß-D-galactopyranosyl)methoxymethyl]methyl-N-[1-(6-methyl)adipyl]glycinamide
[TG(13Å)], and
N-[tris-O-(3,6,9-trioxaundecanyl-ß-D-galactopyranosyl)methoxymethyl]methyl-N-[1-(6-methyl)adipyl]glycinamide
[TG(20Å)] will be described in detail
elsewhere.17A
TG(20Å)C was synthesized, as will be described in detail elsewhere
(E.A.L.B., H.B., J.H.V.B., T.J.C.V.B, unpublished results).
Isolation of Kupffer and Parenchymal Cells
Male Wistar rats
(
250 g body wt each) were anesthetized by
peritoneal injection of 20 mg sodium pentobarbital. Parenchymal liver
cells were isolated after a 20-minute perfusion of the liver with
0.05% collagenase type IV at 37°C according to the method of
Seglen,18 modified as previously described.19
After perfusion, parenchymal and Kupffer cells were purified by
differential centrifugation and counterflow elutriation, as described
in detail elsewhere.20 The purity of the Kupffer cells was
>95%, as judged by peroxidase staining (0.1% 3,3'-diaminobenzidine
in 0.05 mol/L Tris-HCl, 7% sucrose, 0.1% (vol/vol) 30%
H2O2, pH=7.4) for 20 minutes at
37°C.
Isolation and Radioiodination of Asialo-orosomucoid and LDL
Human orosomucoid was isolated and subsequently
desialylated enzymatically, as described previously.21
Human LDL (1.024<d<1.063) was isolated by density-gradient
ultracentrifugation, according to Redgrave et al22 (see
also Bakkeren et al23 ). The purity of LDL was monitored by
PAGE analysis, by agarose-gel electrophoresis (0.8%), and by
particle-size electrophoresis. LDL contained <0.06% apoE and >99%
apoB. The mean diameter was 23±1 nm, whereas <1% of the mass was
recovered in particles >40 nm. LDL was lactosylated by reductive
alkylation of the lysines, as described previously.24 Both
lipoproteins were radiolabeled with carrier-free
[125I]NaI by the iodine monochloride method of McFarlane
as modified by Bilheimer et al.25
In Vitro Binding Studies
Displacement of
125I-asialo-orosomucoid (ASOR)
binding to parenchymal liver cells by unlabeled ASOR or by cluster
galactosides [TG(4Å), TG(9Å), TG(10Å), TG(13Å),
and TG(20Å)] was
determined as follows. Rat parenchymal liver cells (1 to
1.5x106 cells, viability >90%) were incubated in
1 mL DMEM containing 2% BSA with 5 nmol/L 125I-ASOR in the
presence or absence of cluster galactosides in eight concentrations
ranging from 1 nmol/L to 1 mmol/L. After the liver cells were incubated
for 2 hours at 4°C under gentle agitation, the medium was removed by
aspiration and the cells were washed twice in 2 mL ice-cold medium
containing 0.2% BSA and once in medium without BSA. Nonspecific
binding was measured in the presence of 100 mmol/L N-acetyl
galactosamine (GalNAc).
Displacement of binding of 125I-lactosylated LDL (125I-Lac-LDL) to Kupffer cells by unlabeled Lac-LDL or by the newly synthesized cluster galactosides [TG(4Å), TG(9Å), TG(10Å), TG(13Å), and TG(20Å)] was determined analogously. Rat Kupffer cells (1 to 1.5x106 cells, viability >90%) were incubated in 1 mL DMEM containing 2% BSA with 5 nmol/L 125I-Lac-LDL in the presence or absence of cluster galactosides at eight concentrations ranging from 1 nmol/L to 1 mmol/L. After the Kupffer cells were incubated for 2 hours at 4°C under gentle agitation, the medium was removed by aspiration and the cells were washed twice with 2 mL ice-cold medium containing 0.2% BSA and once with medium without BSA. Nonspecific binding was measured in the presence of 100 mmol/L GalNAc.
Protein contents were determined according to the method of Lowry, with BSA as the standard. Displacement binding data were analyzed according to a single-site model with a computerized nonlinear fitting program (Graph-Pad) to calculate the inhibition constant, Ki.24
Animal Experiments
Male Ry-Wistar rats (250 to 300 g each)
were anesthetized
with ether, and a 300-µL blood sample was collected by orbital
puncture. Subsequently, 500 µL NaPi buffer (10 mmol/L, pH
7.4) containing 150 mmol/L NaCl (PBS) or PBS containing TGlc(4Å)C (560
µg) or TG(20Å)C (56, 180, or 560 µg) was injected in the vena
penis, and blood samples (300 µL) were taken at the indicated times
by orbital puncture (three animals per treatment). After sampling,
blood samples were centrifuged for 5 minutes at 1500g; the
serum was collected and stored for further analysis. After the last
puncture, the rats were killed and exsanguinated. The serum was assayed
for the concentration of total cholesterol with a CHOD-PAP kit
(Boehringer Mannheim). The sera obtained at t=24 hours were subjected
to density-gradient ultracentrifugation in NaCl-KBr buffer for 22 hours
at 150 000g. The gradient was subsequently subdivided
according to density by aspiration of 300-µL fractions, starting from
the bottom of the tube; very-low-density lipoprotein (VLDL,
d<1.006), intermediate-density lipoprotein (IDL,
1.006<d<1.019), LDL (1.024<d<1.055), and HDL
(1.063<d<1.21 mg/mL) fractions were isolated from the
density gradient. Since the densities of rat lipoproteins differ
slightly from those of human lipoproteins, the isolation procedure of
Redgrave was slightly adapted according to Bakkeren et
al.23 The purity of the isolated lipoprotein fractions was
verified by PAGE analysis and was always >95%. The lipoprotein
fractions were assayed for total cholesterol content, as described
below. TG(20Å)C did not interfere with the cholesterol assay in the
absence or the presence of cholesterol esterase.
Liver Uptake of Lipoprotein-TG(20Å)C Complexes
Male
Wistar rats (250 to 300 g body wt each) were
anesthetized by injection of 15 to 20 mg IP sodium pentobarbital. The
abdomen was opened, and the complexes of 125I-LDL or
125I-HDL (50 µg apolipoprotein in 500 µL PBS) and
TG(20Å)C, which were prepared by incubation of the lipoprotein
with 50 µg TG(20Å)C for 30 minutes at 20°C, were injected
into the inferior vena cava. Five minutes after injection, a liver
lobule was tied off, excised, weighed, and counted for radioactivity.
The excised liver tissue amounted to <15% of the total liver mass.
The liver uptake of the injected compound was corrected for
radioactivity in serum assumed to be entrapped in the tissue at the
time of sampling (85 µL/g fresh wt).26
Bile Sampling
Bile was collected from unrestrained
3-month-old Wistar rats, as
reported previously.27 Rats received tap water and
standard chow ad libitum and were equipped with permanent catheters in
the bile duct, duodenum, and heart. To maintain an intact enterohepatic
circulation, the bile duct and duodenum catheters were connected
immediately after surgery. Rats were allowed to recover for 1 week. At
the start of the experiment, 560 µg TG(20Å)C (dissolved in 500
µL
PBS) or 500 µL PBS was introduced within 1 minute through the heart
catheter. The bile-duct catheter was then connected to a fraction
collector, and the bile was collected for 48 hours. After the bile
acids were extracted from the cholesterol–cholesterol ester fraction
at pH 7.0, according to Bligh and Dyer,28 the aqueous
layer was analyzed for bile-acid content and the organic phase was
analyzed for total cholesterol content as described below.
Bile-Acid and Total Cholesterol Contents
Total cholesterol
content of the sera was determined
colorimetrically, in duplicate, with a CHOD-PAP kit (Boehringer
Mannheim). The content of bile acids in the bile was determined
colorimetrically with a Sterognost-3 PHO kit from Nycomed with
sodium taurocholate (Sigma) as the standard.
Statistical Analysis
The time curves for the effect of
TG(20Å)C administration on
the serum cholesterol level were analyzed statistically on the basis of
the significance of the difference between four treatment groups and of
a potential effect of time on the cholesterol content for the treatment
groups by repeated-measures ANOVA after correction for missing values
(SPSS/PC+). The effect of TG(20Å)C on the cholesterol
content of the various lipoprotein fractions from serum (Fig 4
)
was
evaluated statistically by one-way ANOVA (SPSS/PC+),
whereas the significance of the differences between means was tested by
unpaired two-way Student's t test.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Affinity and Specificity of Galactose-Terminated Triantennary Cluster Galactosides for the Hepatic Asialoglycoprotein Receptor
The affinity of the synthesized cluster galactosides (see Fig 1A
for structures) for the hepatic asialoglycoprotein
receptor was determined by use of competition studies of
125I-ASOR binding to parenchymal liver cells. The affinity
of the cluster galactoside for the asialoglycoprotein receptor
increased dramatically with elongation of the spacer connecting the
terminal galactosyls with the branching point of the dendrite (Fig
2A).
TG(20Å), provided with a 20-Å spacer, displayed an
Ki of 190 nmol/L, whereas TG(4Å), with a
4Å spacer, was only marginally capable of inhibiting
125I-ASOR binding (Ki=390
µmol/L). The cluster galactosides with intermediate spacer length,
TG(9Å), TG(10Å), and TG(13Å), exhibited intermediate
affinities
(Ki=19, 1.2, and 10 µmol/L,
respectively) for the asialoglycoprotein receptor. As a control,
competition studies of 125I-ASOR binding by unlabeled ASOR
were performed. In agreement with previous studies, the affinity of
ASOR for the asialoglycoprotein protein receptor was in the low
nanomolar range, with an Ki of 6.46±1.75
nmol/L.32 33
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), or a cluster
galactoside: TG(4Å) (
), TG(9Å) (
), TG(10Å)
(
),
TG(13Å) (
), or TG(20Å) (
). Bottom, Kupffer cells were
incubated in 5 nmol/L125I-Lac-LDL in the absence or
presence of unlabeled Lac-LDL(
In addition to the
asialoglycoprotein receptor on the parenchymal liver
cell, the liver contains a second galactose-recognizing receptor: the
galactose-fucose receptor on the Kupffer
cell.24 29 30 To
assess the cellular specificity of the synthesized cluster
galactosides, we have determined the affinity of the galactosides for
the competing galactose-fucose receptor on Kupffer cells by use of
competition studies of 125I-Lac-LDL binding to this
receptor. Lac-LDL has been established to be specifically recognized by
the fucose-galactose receptor on Kupffer cells.30
125I-Lac-LDL binding to Kupffer cells could be inhibited up
to 78% by excess unlabeled Lac-LDL at an
Ki of 1.15±0.30 nmol/L (Fig 2B
). In
contrast, none of the compounds was capable of displacing
125I-Lac-LDL binding from Kupffer cells at concentrations
of up to 400 nmol/L.
Effect of TG(20Å)C on the Cholesterol Concentration of Rat
Serum
The galactoside with the highest affinity for the
asialoglycoprotein receptor TG(20Å) has been derivatized with a
cholesterol moiety, yielding TG(20Å)C (Fig 1B) (E.A.L.B.,
H.B.,
J.H.V.B., T.J.C.V.B., unpublished results), and the biological activity
of the compound has been evaluated in the rat. Although the rat is not
the most appropriate model for evaluating the hypocholesterolemic
activity of TG(20Å)C in terms of serum lipoprotein profile and
cholesterol metabolism, utilization of the rat enables a direct
comparison with earlier studies on the hypocholesterolemic activity of
TG(4Å)C and
mono-gal-chol.12 13 14 15 16 17
The effect of an
intravenous bolus injection of TG(20Å)C on the total serum cholesterol
content in rats is shown in Fig 3. A dose-dependent
decrease of cholesterol level was observed after injection of
TG(20Å)C. A slight initial decrease of the cholesterol level was
induced, even at a dose of 56 µg. A dose of 560 µg TG(20Å)C 11
hours after injection reduced the serum cholesterol level by 45%.
Repeated-measures ANOVA of the concentration-time curves for the four
treatment groups revealed that there were statistically significant
differences between these curves (P<.001). In subsequent
analyses, it was found that the curves for the groups of rats treated
with 180 and 560 µg TG(20Å)C differed significantly from the control
group (P=.002 and P=.004, respectively),
whereas
the curve for 56 µg TG(20Å)C differed only marginally from that of
the control group (P=.076). In addition, a clear-cut and
comparable effect of time on the serum cholesterol levels was noticed
for all treatment groups (P<.01). The persistence of this
reduction was remarkable. Twenty-four hours after administration of the
agent, serum levels still had not reached control values. The effect of
TGlc(4Å)C, the cholesterol derivative of a triantennary cluster
glucoside, was studied as a control for a potential nonspecific effect
of cholesterylated cluster galactosides. However, a bolus injection of
560 µg TGlc(4Å)C did not affect the level of total serum cholesterol
over a 24-hour period after injection (2.28±0.2 mmol/L).
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), or 560
µg TG(20Å)C (
The
levels of the various individual lipoprotein fractions were
measured 24 hours after administration of PBS or TG(20Å)C. In view of
the predominant contribution of HDL cholesterol to the total serum
cholesterol level in rats (65%), it was anticipated that the level
of HDL must be affected by administration of TG(20Å)C. Indeed,
the HDL level was reduced in a dose-dependent manner to a maximum of
35% at a dose of 560 µg (P=.0066 by one-way ANOVA) (Fig
4). The LDL level tended to decrease by 25% to 30%
(P=.036). Surprisingly, administration of TG(20Å)C tends
to
enhance the VLDL level (P=.07). Serum samples of the
TG(20Å)C-treated animals did not exhibit any sign of hemolysis.
Effect of TG(20Å)C on Biliary Secretion in Rats
Subsequently, we investigated whether administration of
TG(20Å)C influenced biliary secretion. TG(20Å)C (560 µg) was
injected into unrestrained rats that were equipped with catheters in
the bile duct, duodenum, and heart; bile was collected for 48 hours,
and the bile flow and amount of bile acid–cholesterol secretion were
determined. The biliary secretion of cholesterol in the
TG(20Å)C-treated rats was identical to that in the control rats (0.41
versus 0.37 µmol/h, respectively) (Fig 5). In
contrast, the secretion of bile acids in the bile was significantly
accelerated, from 72.9±23 to 152±22 µmol per 2 hours during
the
first 2 hours after injection of TG(20Å)C (n=3, P<.05
by
Student's t test, Fig 5). After 2 hours, the
rate of
bile-acid secretion in the TG(20Å)C-treated rats stabilized at control
values, ie, 9.5±2.8 and 7.7±2.0 µmol/h, respectively. The
biliary
flow (Fig 5) was not affected by injection of TG(20Å)C
(0.75 versus
0.70 mL/h, n=3).
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Effect of Loading of 125I-HDL and 125I-LDL
With TG(20Å)C on the Liver Uptake of Lipoproteins
The liver
uptake of native 125I-HDL 5 minutes after
injection was low (1.9±1.4% of the injected dose; see
Table). However, incubation of 50 µg
125I-HDL with 15 µg TG(20Å)C markedly stimulated the
hepatic uptake of the lipoprotein to 33.6±1.3% of the injected dose
(P<.001 by Student's t test). Preincubation of
50 µg 125I-LDL with 15 µg TG(20Å)C also caused a
significant increase in liver association of the lipoprotein from
1.7±0.4% to 20.1±1.2% of the injected dose (P<.002
by
Student's t test). To verify whether hepatic
galactose-recognizing receptors are involved in the increased
up-take of TG(20Å)C-lipoprotein complexes, we studied the effect of
preinjection of GalNAc on the liver uptake of these complexes. The
hepatic uptake of both TG(20Å)C–125I-HDL and
TG(20Å)C–125I-LDL appeared to be almost completely
inhibited by an excess of GalNAc (89% and 81% inhibition,
respectively; P<.002 by Student's t test).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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We previously reported that lipoprotein catabolism can be enhanced by administration of lipophilized galactose-terminated galactosides, which induce uptake of lipoproteins by hepatic galactose-recognizing receptors.13 14 15 However, prolonged infusion of relatively high doses of these galactosides was required to lower the blood cholesterol content significantly. We rationalized that the low potency may be caused by the low affinity and specificity of the galactose-terminated cluster galactoside for the hepatic asialoglycoprotein receptor. On the basis of previous studies of the prerequisites for high-affinity recognition by this receptor,31 32 33 and by use of molecular modeling, new triantennary cluster galactosides have been designed and synthesized.17A In the present study, we demonstrate that the affinity of these new galactosides for the asialoglycoprotein receptor is markedly increased as a result of proper spacing of the terminal galactose units within a triantennary cluster galactoside. TG(20Å), in which the terminal galactose moieties were spaced 20 Å from the branching point of the dendrite, possessed a 2000-fold higher affinity (Ki=190 nmol/L) than a previously used cluster galactoside, TG(4Å)12 13 14 (Ki=390 µmol/L). TG(9Å), TG(10Å), and TG(13Å) displayed intermediate affinities for the asialoglycoprotein receptor. Remarkably, TG(10Å) had a slightly higher affinity for the asialoglycoprotein receptor than TG(9Å) or TG(13Å). This difference probably results from the higher hydrophobicity of the propyl moiety of TG(10Å) compared with the ethylene glycol units TG(9Å) and TG(13Å), as has been suggested previously for other triantennary galactoside substrates of the asialoglycoprotein receptor.33
In contrast, elongation of the spacers within a cluster galactoside from 4 to 20Å did not influence the affinity for the galactose-fucose receptor on the Kupffer cell, which also recognizes galactose-terminated galactosides. Hence, it can be concluded that not only the affinity but also the specificity for the asialoglycoprotein receptor compared with the galactose-fucose receptor is dramatically enhanced upon elongation of the spacer. In view of the affinity of ASOR for the asialoglycoprotein receptor, an additional 25-fold gain in affinity and specificity may be achieved for synthetic cluster galactosides. However, the affinity of TG(20Å) for the asialoglycoprotein receptor, which is 2000-fold higher than that of TG(4Å), may indicate that the cholesterol-lowering activity of a cholesterylated galactoside correlates with an increase in the affinity of the galactoside for the asialoglycoprotein receptor; further improvement in affinity may lead to direct clearance of the compound before the compound can accumulate in the lipoproteins. Thus, we have derivatized the most selective galactoside, TG(20Å), with cholesterol. The resulting compound, TG(20Å)C, is an amphiphilic compound. Previous study has demonstrated that TG(20Å)C incorporates spontaneously into lipoproteins in incubation with serum17A and with isolated lipoproteins.34 Subsequently, we evaluated the physiological activity of TG(20Å)C in the rat. Intravenous bolus injection of TG(20Å)C into rats resulted in a significant dose-dependent decrease of the serum cholesterol concentration. A maximal decrease (45%) of the serum cholesterol concentration was observed after a single injection of only 560 µg TG(20Å)C; its hypocholesterolemic potency was at least 30-fold higher than that of the previously developed compound TG(4Å)C.12 In contrast to TG(4Å)C, application of TG(20Å)C did not require an infusion protocol and did not lead to hemolysis at therapeutic doses.12 Even intravenous injection of 6 mg TG(20Å)C/kg into rats was tolerated well. The decrease in the serum cholesterol level persisted for at least 24 hours, possibly reflecting the low rate of de novo synthesis of HDL in the rat. Alternatively, this decrease may arise from storage of TG(20Å)C in and sustained release from a hydrophobic compartment in the rat (ie, cell membranes).
Further analysis of the serum lipoprotein profile of both TG(20Å)C-treated and untreated control rats showed that the decrease in the level of total serum cholesterol can be attributed mainly to an interaction of TG(20Å)C with HDL and, to a lesser extent, with LDL. However, before extrapolating these in vivo results in rats to the human situation, one should realize that the ratio of LDL-cholesterol to HDL-cholesterol in the rat is approximately 15-fold lower than in humans (0.2 and 3.0, respectively). The rat evidently is not the most appropriate species for studying the in vivo LDL-lowering activity of TG(20Å)C. Further studies must therefore be performed to investigate the hypocholesterolemic activity of TG(20Å)C in an animal more comparable to the human species in terms of lipoprotein profile and cholesterol metabolism (ie, the Watanabe heritable hyperlipidemic rabbit or cholesterol-fed hamster). At first glance, accelerating the catabolism of HDL seems to be undesirable because it might result in a more atherogenic plasma lipoprotein profile. However, a compound that selectively enhances the hepatic uptake of HDL may concomitantly stimulate the HDL-mediated reverse cholesterol transport from the periphery to the liver, which, of course, is beneficial. In this respect it needs to be verified whether long-term administration of TG(20Å)C will affect the reverse cholesterol transport of HDL, the rate of de novo synthesis of HDL and, consequently, the lipoprotein profile.
The high potency of the compound to direct lipoproteins to the
liver is illustrated by the amount of TG(20Å)C molecules that is
required for the removal of one molecule of HDL from the bloodstream.
If we assume that the total HDL pool of the rat is 80 nmol, the
highest dose of TG(20Å)C (560 µg=0.3 µmol per rat)
corresponds to
4 moles TG(20Å)C per mole HDL. Incorporation of 4 moles of
TG(20Å)C
per HDL apparently suffices to induce efficient removal of the
lipoprotein from the bloodstream. Further study has demonstrated that
the hepatic uptake of 125I-HDL and 125I-LDL was
significantly increased upon incubation of these lipoproteins in
TG(20Å)C before injection into the rat. The TG(20Å)C-induced
stimulation of the liver uptake of 125I-HDL and
125I-LDL could be prevented by preinjection of an excess of
GalNAc, which suggests the involvement of galactose-recognizing
receptors in the TG(20Å)C-induced liver uptake of HDL and LDL. Recent
studies with premixed TG(20Å)C-LDL and TG(20Å)C-HDL clearly
identified the parenchymal liver cell to be responsible for the hepatic
uptake of TG(20Å)C–125I-HDL and
TG(20Å)C–125I-LDL complexes.34 This is
rather surprising, since previously developed cholesterylated
galactosides TG(4Å)C and mono-gal-chol induced uptake of LDL by the
galactose-fucose receptor24 29 30 on the
Kupffer
cell.13 15 We therefore suggest that the
TG(20Å)C-induced
reduction in the serum cholesterol level arises from an increased
hepatic uptake of lipoproteins by the asialoglycoprotein receptor on
parenchymal liver cells. An additional indication that parenchymal
cells are involved in the increased clearance of cholesterol from the
circulation was obtained from the effect of TG(20Å)C on biliary
secretion in the rat. During the first 2 hours after administration of
TG(20Å)C, the secretion of bile acids was two times normal levels
(P<.05), whereas the biliary flow and the secretion of
cholesterol remained constant. This suggests that TG(20Å)C-induced
hepatic uptake of cholesterol is rapidly shunted into the bile-acid
pathway, a route morphologically linked to the parenchymal cell. The
reduction in serum cholesterol (15 µmol), however, is too small to
fully account for the observed increment in the secretion of bile
acids. Apparently, TG(20Å)C also mobilizes bile acids from the
bile-acid pool. A more direct study of the effect of TG(20Å)C on the
biliary secretion profile is required to unravel this phenomenon.
In conclusion, the present data show that TG(20Å)C is a promising and potent serum cholesterol–lowering agent. In rats, TG(20Å)C principally induces hepatic uptake of HDL, thereby stimulating reverse cholesterol transport. Further study of TG(20Å)C or analogues on the lipid metabolism in an animal model that is more comparable with humans in terms of lipoprotein profile and cholesterol metabolism will reveal whether TG(20Å)C is also capable of inducing significant hepatic uptake of LDL. Administration of lipoprotein uptake enhancers, such as TG(20Å)C, involves a completely new approach to treatment of hypercholesterolemia. Its therapeutic activity does not depend on the presence of functional LDL receptors, as do conventional therapies based on HMG-CoA reductase or bile-acid sequestrants. Therefore, we envision use of a therapy involving TG(20Å)C or analogues to be a promising alternative for those patients who do not respond or who respond insufficiently to the aforementioned therapies.
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Acknowledgments |
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This work was supported by a grant from the Dutch Heart Foundation (42.005). Dr E.A. Van de Velde is gratefully acknowledged for his help in statistical analysis.
Received June 2, 1994; revision received September 8, 1994; accepted October 2, 1994.
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